A lifetime window into lipid dynamics in Alzheimer’s disease

Cellular lipids play crucial roles in the cell, including in energy storage, the formation of cellular membranes, and in signaling and vesicular trafficking. To understand the functions and characteristics of lipids within cells, various methods to image lipids have been established. In this Commentary, we discuss the four main types of molecular probes that have significantly contributed to our understanding of the cell biology of lipids. In particular, genetically encoded biosensors and antibodies will be discussed, and how they have been used extensively with traditional light and electron microscopy to determine the subcellular localization of lipids and their spatial and temporal regulation. We highlight some of the recent studies that have investigated the distribution of lipids and their ability to cluster using super-resolution and electron microscopy. We also examine methods for analyzing the movement and dynamics of lipids, including single-particle tracking (SPT), fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). Although the combination of these lipid probes and the various microscopic techniques is very powerful, we also point out several potential caveats and limitations. Finally, we discuss the need for new probes for a variety of phospholipids and cholesterol.

Lipid dynamics and protein-lipid interactions in rat colonic epithelial cell basolateral membranes

In this dissertation, molecular interactions and changes imposed by nano-scale structures on phospholipid vesicles were investigated. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) large unilamellar vesicles (LUVs) were used as the model system. Understanding changes of the bilayer structure, interfacial properties, lipid dynamics, and self-assembly, allows bridging relationships between biological cell membrane structure and dynamics to cellular functionalities. For example, membrane curvature changes are linked to membrane protein functions, although the exact mechanisms of control are not yet understood. Moreover, the knowledge gained from vesicle models allows exploring novel strategies for drug delivery applications. To achieve this, DOPC LUVs were synthesized and characterized by a combination of Dynamic Light Scattering (DLS), cryo-Transmission Electron Microscopy (cryo-TEM), Small-Angle X-Ray, and Neutron Scattering (SAXS, SANS). In combination with the lipid dynamics investigated by Neutron Spin-Echo spectroscopy (NSE) and Nuclear Magnetic Resonance (NMR) relaxometry, several new insights related to vesicle self-assembly and control of structure and dynamics were revealed. Lipid dynamics investigated by NSE showed the first experimental evidence for confined lipid dynamics at short Fourier time (<3>ns) with a mean squared displacement t 0.26. DOPC vesicles in the presence of a hyperosmotic NaCl gradient showed transformation to multilamellar structures and size changes. Size variation showed abrupt changes at certain concentrations as a result of new bilayer formation. Membrane rigidity explored by NSE showed increasing rigidity with increasing external NaCl. Small drug molecules interacting with DOPC vesicles showed different effects on the lipid bilayer structure when introduced in solution-phase and directly inserted into the lipid layer at the beginning of vesicle synthesis. Acetaminophen showed the softening of lipid bilayer by NSE similar to previous studies using Ibuprofen and Aspirin. Interactions between DOPC LUVs and N-alkyl-PEO polymers presented a new way to transform LUVs from unilamellar to multilamellar vesicles. These multilamellar vesicles had a decreased membrane rigidity which is useful for transdermal drug delivery. Lastly, Non-ionic Amphiphilic Alternating Polymers (NAAPs) showing promising solution properties were characterized. They showed LCST behavior close to physiological temperature with high tunability. In the presence of NAAPs, DOPC vesicles showed no significant size changes. However, susceptibility to electron beam/radiation damage appeared to be enhanced.

The objective of this study was to examine the hypothesis that abdominal and gluteal adipocyte turnover, lipid dynamics, and fibrogenesis are dysregulated among insulin-resistant (IR) compared with insulin-sensitive (IS) adolescents with obesity. Seven IS and seven IR adolescents with obesity participated in a 3-h oral glucose tolerance test and a multi-section magnetic resonance imaging scan of the abdominal region to examine body fat distribution patterns and liver fat content. An 8-week 70% deuterated water (2 H2 O) labeling protocol examined adipocyte turnover, lipid dynamics, and fibrogenesis in vivo from biopsied abdominal and gluteal fat. Abdominal and gluteal subcutaneous adipose tissue (SAT) turnover rates of lipid components were similar among IS and IR adolescents with obesity. However, the insoluble collagen (type I, subunit α2) isoform measured from abdominal, but not gluteal, SAT was elevated in IR compared with IS individuals. In addition, abdominal insoluble collagen Iα2 was associated with ratios of visceral-to-total (visceral adipose tissue + SAT) abdominal fat and whole-body and adipose tissue insulin signaling, and it trended toward a positive association with liver fat content. Altered extracellular matrix dynamics, but not expandability, potentially decreases abdominal SAT lipid storage capacity, contributing to the pathophysiological pathways linking adipose tissue and whole-body IR with altered ectopic storage of lipids within the liver among IR adolescents with obesity.

As an eminent ambassador of STEM and renowned NMR spectroscopist, Frances Separovic is an internationally famous name, but could it also be a valuable membrane-active peptide sequence? Her name has been used as an amino acid sequence (FS), successfully synthesised, oxidised, and put into contact with membrane models to investigate any serendipitous activity. The 3D structure of the cyclic FS was determined in dodecylphosphocholine (DPC) micelles by solution NMR spectroscopy. FS displayed a twisted bend separating a helical stretch and an unstructured segment. Using solid-state NMR spectroscopy, the effect of FS on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS) lipid bilayers was studied. FS did not strongly disturb the neutral membrane surface but likely inserted into their hydrophobic core without a strong effect on the lipid dynamics, while perturbation of the negatively charged membranes remained at the headgroup interface with a strong effect on the lipid dynamics. This study demonstrated that FS is a candidate for discovering potential future therapeutic activities.

Methanol Accelerates DMPC Flip-Flop and Transfer: A SANS Study on Lipid Dynamics

Infections caused by many virulent bacterial strains are triggered by the release of pore forming toxins (PFTs), which form oligomeric transmembrane pore complexes on the target plasma membrane. The spatial extent of the perturbation to the surrounding lipids during pore formation is relatively unexplored. Using all-atom molecular dynamics simulations, we investigate the changes in the structure and dynamics of lipids in a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayer in the presence of contrasting PFTs. Cytolysin A (ClyA), an α toxin with its inserted wedge shaped bundle of inserted α helices, induces significant asymmetry across the membrane leaflets in comparison with α hemolysin (AHL), a β toxin. Despite the differences in hydrophobic mismatch and uniquely different topologies of the two oligomers, perturbations to lipid order as reflected in the tilt angle and order parameters and membrane thinning are short ranged, lying within ∼2.5 nm from the periphery of either pore complex, and commensurate with distances typically associated with van der Waals forces. In contrast, the spatial extent of perturbations to the lipid dynamics extends outward to at least 4 nm for both proteins, and the continuous survival probabilities reveal the presence of a tightly bound shell of lipids in this region. Displacement probability distributions show long tails and the distinctly non-Gaussian features reflect the induced dynamic heterogeneity. A detailed profiling of the protein-lipid contacts with tyrosine, tryptophan, lysine and arginine residues shows increased non-polar contacts in the cytoplasmic leaflet for both PFTs, with a higher number of atomic contacts in the case of AHL in the extracellular leaflet due to the mushroom-like topology of the pore complex. The short ranged nature of the perturbations observed in this simple one component membrane suggests inherent plasticity of membrane lipids enabling the recovery of the structure and membrane fluidity even in the presence of these large oligomeric transmembrane protein assemblies. This observation has implications in membrane repair processes such as budding or vesicle fusion events used to mitigate PFT virulence, where the underlying lipid dynamics and fluidity in the vicinity of the pore complex are expected to play an important role.

Recent years have seen the development of multiple technologies to investigate, with great spatial and temporal resolution, the dynamics of lipids in cellular and model membranes. One of these approaches is the combination of far-field super-resolution stimulated-emission-depletion (STED) microscopy with fluorescence correlation spectroscopy (FCS). STED-FCS combines the diffraction-unlimited spatial resolution of STED microscopy with the statistical accuracy of FCS to determine sub-millisecond-fast molecular dynamics with single-molecule sensitivity. A unique advantage of STED-FCS is that the observation spot for the FCS data recordings can be tuned to sub-diffraction scales, i.e. <200nm in diameter, in a gradual manner to investigate fast diffusion of membrane-incorporated labelled entities. Unfortunately, so far the STED-FCS technology has mostly been applied on a few custom-built setups optimised for far-red fluorescent emitters. Here, we summarise the basics of the STED-FCS technology and highlight how it can give novel details into molecular diffusion modes. Most importantly, we present a straightforward way for performing STED-FCS measurements on an unmodified turnkey commercial system using a time-gated detection scheme. Further, we have evaluated the STED-FCS performance of different commonly used green emitting fluorescent dyes applying freely available, custom-written analysis software.

Continuous fluorescence microphotolysis of anthracene-labeled phospholipids in membranes. Theoretical approach of the simultaneous determination of their photodimerization and lateral diffusion rates

Understanding dynamic and complex interaction of biological membranes with extracellular matrices plays a crucial role in controlling a variety of cell behavior and functions, from cell adhesion and growth to signaling and differentiation. Tremendous interest in tissue engineering has made it possible to design polymeric scaffolds mimicking the topology and mechanical properties of the native extracellular microenvironment; however, a fundamental question remains unanswered: that is, how the viscoelastic extracellular environment modifies the hierarchical dynamics of lipid membranes. In this work, we used aqueous solutions of poly(ethylene glycol) (PEG) with different molecular weights to mimic the viscous medium of cells and nearly monodisperse unilamellar DMPC/DMPG liposomes as a membrane model. Using small-angle X-ray scattering (SAXS), dynamic light scattering, temperature-modulated differential scanning calorimetry, bulk rheology, and fluorescence lifetime spectroscopy, we investigated the structural phase map and multiscale dynamics of the liposome–polymer mixtures. The results suggest an unprecedented dynamic coupling between polymer chains and phospholipid bilayers at different length/time scales. The microviscosity of the lipid bilayers is directly influenced by the relaxation of the whole chain, resulting in accelerated dynamics of lipids within the bilayers in the case of short chains compared to the polymer-free liposome case. At the macroscopic level, the gel-to-fluid transition of the bilayers results in a remarkable thermal-stiffening behavior of polymer–liposome solutions that can be modified by the concentration of the liposomes and the polymer chain length.

The lipid core of a biological membrane requires a certain degree of structural rigidity, but it must also be sufficiently fluid to permit lateral movement of the constituent lipids and embedded proteins. Ectotherms can counteract the ordering effects of reduced temperature by changing the structure of their membranes, a process known as homeoviscous adaptation (1). Although the content of unsaturated fatty acids in the membranes of ectothermic animals is generally known to increase in response to cold (2), no clear and direct relationship between unsaturated fatty acids and membrane fluidity has been established in marine organisms. For example, phospholipid molecular species containing docosahexaenoic acid (22:6 3) are believed to be important in controlling finfish membrane fluidity (3–6), but a direct correlation between 22:6 3 and membrane fluidity has not been found (4, 5, 7, 8). In contrast, we show here a simple but very strong relationship between fluidity and a single polyunsaturated fatty acid, eicosapentaenoic acid (20:5 3), in gill membranes from a marine bivalve mollusc, the sea scallop Placopecten magellanicus. Phospholipids are the main structural elements of biological membranes, and their physical characteristics are key determinants of membrane structure and function. Many vital cell activities that depend on the optimal functioning of membranes are therefore sensitive to the chemistry of the membrane lipids (9) and to environmental conditions, such as temperature and pressure, that perturb the phase behavior and dynamics of lipids in membranes (10). Under extreme or variable conditions, organisms can exploit the tremendous chemical diversity among membrane lipids to defend the physical properties of the membrane (10). Thus in ectotherms, where changes in temperature cause important membrane perturbations, the usual adaptive response includes a modification of lipid composition (11). Sessile animals living in Newfoundland waters must maintain membrane structure and function in the face of extreme cold in deep waters (as low as 1.4°C) or seasonally highly variable conditions in surface waters (as much as 22°C in 6 months) (12). In the present study, we exposed sea scallops to a 10°C decrease in temperature for up to 3 weeks and then examined the relationship between the fatty acid composition of branchial phospholipids and membrane fluidity. Vesicles were prepared from the gills of scallops acclimated to temperatures of 15 and 5°C. After three weeks of thermal acclimation, the structural order of the phospholipids was measured by electron spin resonance (ESR) spectroscopy at five temperatures (0–20°C) that span the physiological range of Placopecten magellanicus (Fig. 1). The vesicles prepared from gills of 5°C-acclimated scallops were significantly (ANCOVA, P 0.03) less ordered than vesicles from 15°C-acclimated scallops. Temperature acclimation had shifted the order parameter curve 1–2°C toward lower assay temperatures, giving a homeoviscous efficacy (13) of 14%. Such a partial adjustment towards an ideal or complete homeoviscous response has also been found in crabs (14) and crayfish (15). In these invertebrates, the costs of perfect compensation may be too high, or the benefits too low. On the other hand, the ESR measurements in this study were made with the spin probe 5-doxyl stearic acid, reflecting the homeoviscous response in the outer region of the purified lipid bilayer. It is possible that the response deeper in the bilayer, in the actual region of alkenyl chain unsaturation, would have been greater (16). Received 30 November 2001; accepted 22 March 2002. 1 Present address: Department of Physiology and Experimental Medicine, The George Washington University Medical Center, Ross Hall Room 402, 2300 Eye Street, NW, Washington, DC 20037. 2 To whom correspondence should be addressed. E-mail: cparrish@ mun.ca Reference: Biol. Bull. 202: 201–203. (June 2002)

The lateral dynamics of proteins and lipids in the mammalian plasma membrane are heterogeneous likely reflecting both a complex molecular organization and interactions with other macromolecules that reside outside the plane of the membrane. Several methods are commonly used for characterizing the lateral dynamics of lipids and proteins. These experimental and data analysis methods differ in equipment requirements, labeling complexities, and further oftentimes give different results. It would therefore be very convenient to have a single method that is flexible in the choice of fluorescent label and labeling densities from single molecules to ensemble measurements, that can be performed on a conventional wide-field microscope, and that is suitable for fast and accurate analysis. In this work we show that k-space image correlation spectroscopy (kICS) analysis, a technique which was originally developed for analyzing lateral dynamics in samples that are labeled at high densities, can also be used for fast and accurate analysis of single molecule density data of lipids and proteins labeled with quantum dots (QDs). We have further used kICS to investigate the effect of the label size and by comparing the results for a biotinylated lipid labeled at high densities with Atto647N-strepatvidin (sAv) or sparse densities with sAv-QDs. In this latter case, we see that the recovered diffusion rate is two-fold greater for the same lipid and in the same cell-type when labeled with Atto647N-sAv as compared to sAv-QDs. This data demonstrates that kICS can be used for analysis of single molecule data and furthermore can bridge between samples with a labeling densities ranging from single molecule to ensemble level measurements.

Numerous changes occur post-mortem in fish, affecting its chemical composition and nutritional quality. In the present paper we describe the effect of storage on ice or at -30 degrees C or -80 degrees C on 10 species of Mediterranean fish. Water and lipid soluble antioxidants, lipid pattern and products of oxidative attack on lipids, proteins and DNA were quantified for 7 consecutive days on homogenates of fish light muscle. The earliest events were oxidation of ubiquinol and vitamin C, which disappeared almost completely within 48 hours. Ubiquinol oxidation gave rise to an initial increase of ubiquinone, which peaked at the second day: thereafter ubiquinone itslef decreased, more rapidly and to a greater extent than vitamin E. The decrease in antioxidants was accompanied by significant oxidative damage to lipids, proteins and DNA. TBARS significantly increased beginning from the third day of storage in all species and were linked to a significant reduction in the n-3 PUFA of triglycerides (TG) and phospholipid fractions (PL). A remarkable elevation of protein carbonyls and 8OHdG occurred approximately 24 hours later than PUFA oxidation. For SOD, GPX and GSH significant depletions occurred for all species only at 6th or 7th day, but the final values were always higher than 50% compared to the initial ones. Deep-freezing of the same species at -30 degrees C and -80 degrees C for up to 12 months did not significantly affect the levels of enzymatic antioxidants, the redox couple GSH/GS-SG, n-3 and n-6 PUFA of TG and PL fractions of the light muscle. The only antioxidants, which at -30 degrees C and -80 degrees C appeared to be degraded after 6 and 12 months were ubiquinol and vitamin C. As expected their degradation was higher at -30 degrees C than at -80 degrees C. In fact the average decrease for ubiquinol at -80 degrees C was 42% at 6 and 12 months respectively, whereas at -30 degrees C the decrease was 61% and 87% For vitamin C the average decrease at -80 degrees C was 36% and 67% at 6 and 12 months respectively, and at -30 degrees C it was 61% and 82%. Vitamin E was considerably more stable than ubiquinol and vitamin C. The relative stability of the antioxidants, with the exceptions of ubiquionols, vitamin C and, to a certain extent, vitamin E, was accompanied by a very limited increase in oxidation products. In addition no significant hydrolysis of TG and PL fractions were observed throughout the storage time. The dynamics of lipid, protein and DNA oxidation is discussed in the light of depletion of the various antioxidant systems.

Dynamics of Lipids in Biomembranes

The phospholipid bilayers are the primary constituents of the membrane in living cells in which lipids are hold together in bilayer leaflets through a combination of different forces into the liquid crystalline (Lα) phase. Despite their thin fragile formations, the phospholipid bilayers are responsible for performing a variety of important tasks in the cells, some of which are carried out directly by the lipid bilayers and some by various integral proteins embedded within the bilayers. There have been continues efforts over the past decades to replicate the compound biophysical properties of living cell membranes in model lipid bilayers. An important question remains unanswered: is it possible to replicate physical properties under “non-equilibrium” conditions as found in cell membranes in model lipid bilayers? In almost all previous studies, the model lipid bilayers were under static conditions – for instance, at zero lateral pressure. However, in living organisms, the cell membranes are involved in continuous (nonequilibrium) exchange and (or) transport of lipid species with the surrounding environment which consequently leads them to experience continuous lateral pressure variations. One suitable in vitro approach is to spatiotemporally control the model lipid bilayers over a time period during which they can be spatially stimulated at a level compatible to that found under in vivo conditions. This can be achieved with high spatiotemporal resolution by making lipids light-dependent through implementation of azobenzene photoswitch in their structures. In this study, a specific azobenzene containing photolipid (AzoPC) is integrated into POPE:POPG bilayers (POPE: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, POPG: 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)) at ~14 mol% to construct a photo responsive model bilayers entitled as photoliposomes. Magic angle spinning solid-state NMR spectroscopy (MAS-NMR) at high field (850 MHz) is the measurement technique of choice by which it is possible to pursue the dynamics (fluidity) of the bulk lipids within the photoliposomes at atomistic resolution. It is shown that the AzoPCs undergo an efficient trans-to-cis isomerization (~85%) within the photoliposomes as the result of UV light absorption, and thermally relax back to the trans state during a period of ~65 h under the MAS measurement conditions. The order parameter measurements based on the C−H dipolar couplings reveal that the non-equilibrium cis-to-trans thermal isomerization impact of AzoPC on the fluidity of the bulk lipid is highly localized – the fluidity perturbations originate from specific order parameter changes in the middle section of the bulk lipid acyl chains. Further 1H NOESY measurements confirm VI the hypothesis that the azoswitch topologies in either cis and trans conformer of the photolipid is the key parameter in localized alteration of the C−H order parameters along the bulk lipid acyl chains. Diacylglycerol kinase (DgkA) from E. coli is an enzyme responsible for the phosphorylation of diacylglycerol to phosphatidic acid, at the expense of adenosine triphosphate. Structurally, DgkA is a homo oligomer composed of three symmetric 14 kDa protomers, each of which has three transmembrane helices and one surface helix. Upon embedding within the photoliposomes, it is shown that DgkA enhances the AzoPC localization impact on the fluidity of the bulk lipids. In this regard, the results of a series of statistical simulations of lipid lateral diffusions along the bilayer leaflets in presence and absence of embedded proteins are accompanied with those of experimentally measured based upon which it is justified that membrane proteins markedly limit lipid lateral diffusions in the bilayers. In case of the DgkA proteo-liposomes with lipid-to-protein ratio of 50, it is estimated that the diffusion coefficient of lipids is above 2-fold lower compared to that of the protein free liposomes. The cis-to-trans AzoPC isomerization and its following consequence in localized alteration of the bulk lipid fluidity is further investigated on the structural dynamics and enzymatic functionality of the embedded DgkA within the proteo-photoliposomes. It is revealed that DgkA structural dynamics are perturbated in a multi-scale, complex manner. The dynamics of residues located in different regions of DgkA changes with the light-induced AzoPC isomerization, but their time courses differ from residue to residue. For example, 29Ala, a residue on the hinge between the surface helix and membrane helix-1, exhibits the steepest time-dependent cross peak intensity changes in time-resolved NCA spectra. The impact of the lasting membrane fluidity perturbation on the enzymatic functionality of the embedded DgkA is subsequently measured which demonstrates a significant variation under cis- and trans-AzoPC conformations within the proteo-photoliposomes.